The goal of the propoposed project is to develop Caenorhabditis elegans whole-animal high-throughput screening assays in 96-well plates that lead to the identification of novel classes of antimicrobial or immune- enhancing compounds. Previous work has shown that many human pathogens infect and kill the nematode C. elegans. The proposed overall strategy is to identify natural or synthetic compounds that cure C. elegans of a persistent bacterial infection. We anticipate that this work will establish approaches that solve some of the main obstacles in current antimicrobial discovery - finding newclasses of compounds, solving the bottleneck of toxicity/efficacy testing, and delivering the drug to the target pathogen. Preliminary results demonstrate that the C. elegans pathogenicity model can be used to assay compounds with antimicrobial activity. A prototype assay for screening for anti-infective or immune enhancing compounds was developed that takes advantage of the fact that the Gram positive human opportunistic pathogen, Enterococcus faecalis, forms a persistent infection in the C. elegans intestine. The assay was used to screen 6000 small molecules and 1224 natural product extracts, leading to the identification of 16 compounds and 9 extracts that were active in the assay. In addition MDR pump inhibitors were shown to act in synergy with antimicrobials in the C. elegans curing assay. The preliminary data indicate that a nematode model will produce hits that are overlooked in a conventional in vitro screen and that the nematode is a useful intermediate between an in vitro screen and testing in mammals. A major goal of the proposed screen development project is to increase the throughput of the C. elegans-E. faecalis whole-animal screening assay to allow screening of hundreds of thousands or millions of compounds and extracts. The C. elegans screening methodology will also be extended to include the Gram negative pathogen Salmonella enterica and adapted to enable the identification of multi-drug resistant (MDR) pump inhibitors. There are three specific aims: 1) Develop a high throughput E. faecalis curing assay in part by automating the scoring of worm viability. 2) Develop a high throughput C. elegans - S. enterica curing assay. 3) Develop a high throughput screen for identifying MDR prump inhibitors in the C. elegans curing assays.